1. Field of the Invention
This invention pertains to the fields of antifungal therapeutics and drug discovery. More specifically, the invention pertains to the use of fungal mutations affecting microtubule stability to identify compounds suitable as antifungal agents.
2. Description of Related Art
The incidence and severity of fungal infections have increased greatly over recent years. Fungal infections of particular concern in recent times include aspergillosis (infection by Aspergillus sp., such as Aspergillus fumigatus), cryptococcosis (infection by Cryptococcus neoformans), zygomycosis (infection by zygomycetes), and candidiasis (infection by Candida sp.). Some of these fungal infections, such as disseminated aspergillus, disseminated cryptococcosis, and zygomycosis, are frequently fatal, especially in a debilitated patient. Furthermore, some fungal infections, such as candidiasis and cryptococcosis, are extremely common in aids patients
Current antifungal therapies typically employ amphotericin B, 5-flucytosine, caspofungin, and/or various azole derivatives such as fluconazole, ketoconazole, and itraconazole. No known form of therapy is effective on all fungal infections, and all of the known therapies carry with them some level of human toxicity. Increasing levels of resistance to known therapies also present a problem. Despite efforts to minimize toxicity and maximize potency of the azoles since the 1970s, amphotericin B, a drug introduced in the 1950s, remains the best choice for many serious mycoses, and, in particular, disseminated mycoses, despite the drug's significant toxicity and problems with resistance and non-availability of an absorbable oral form for long-term maintenance. Attempts to encapsulate amphotericin B into liposome vesicles to diminish toxicity have proven only moderately successful.
During mitosis, the mitotic spindle undergoes a complex series of transitions. At each mitotic cell division, the spindle assembles, forms attachments to the chromosomes, orients itself properly within the cell, and carries out chromosome segregation, before disassembling again. Proper spindle assembly and function involves coordination of many cellular events including the creation and control of at least three distinct types of microtubules: kinetochore, polar, and astral microtubules. Microtubules mediate a series of movements that culminate in chromosome segregation, including migration of the nucleus to the neck between the mother and daughter cells, assembly of a bipolar spindle, translocation of the spindle through the neck, and elongation of the spindle. This set of molecular events results in chromosome to pole movement (anaphase a) and separation of spindle poles (anaphase b).
As cells enter mitosis, cytoplasmic microtubules depolymerize and the mitotic spindle forms. During mitosis, the dynamic spindle microtubules undergo specific depolymerization events. Perturbing either the depolymerization or polymerization of microtubules in mitosis, either with drugs or by genetic lesions, interferes with mitotic progression, sometimes leading to arrest in proliferation.
Some agricultural fungicides have also been reported as having antimitotic modes of action. For instance, benomyl, a major agricultural fungicide, acts as a microtubule destabilizer in fungi. Similarly acting compounds include carbendazim (an active breakdown product of benomyl), and thiabendazole (an agricultural and veterinary fungicide). However, these fungicides are generally not very specific. Furthermore, the potential health hazards of some of these fungicides, such as benomyl, have created concern and are the target of investigations (see, e.g., Watterson, J. of Public Health Medicine, 16:141-144(1994).
Accordingly, the discovery of antifungal agents with novel mechanisms of action would be highly desirable.